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Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.
Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 °C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial "tubes" are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.
Blood flow throughout the brain is regulated by the coordination of vasodilation among cerebral arteries and arterioles in vascular networks1. Endothelial cells lining cerebral resistance arteries command changes in vascular diameter as needed to meet the metabolic demand of neurons1,2,3. In particular, during endothelium-derived hyperpolarization (commonly known as EDH), intracellular Ca2+ ([Ca2+]i) and electrical signaling in endothelial cells coordinate vasodilation among endothelial cells and their surrounding smooth muscle cells through gap junctions for arterial relaxation4. Physiological initiation of EDH sequentially entails stimulation of Gq-coupled receptors (GPCRs), an increase in [Ca2+]i, and activation of endothelial small- and intermediate-Ca2+-activated K+ (SKCa/IKCa) channels to hyperpolarize cerebral endothelial membrane potential (Vm)5,6,7. Thus, the intimate relationship of endothelial [Ca2+]i and Vm is integral to blood flow regulation and indispensable to cardio- and cerebrovascular function6,8. Throughout the broader literature, numerous studies have reported the association of vascular endothelial dysfunction with the development of chronic diseases (e.g., hypertension, diabetes, heart failure, coronary artery disease, chronic renal failure, peripheral artery disease)9,10, indicating the significance of studying endothelial function in physiological as well as pathological conditions.
Vascular endothelium is integral to the production of hyperpolarization, vasodilation, and tissue perfusion and thus, examination of its native cellular properties is crucial. As a general study model, preparation of the mouse arterial endothelial tube model has been published before for skeletal muscle11,12, gut13, lung14, and recently for the brain6. Studies of simultaneous [Ca2+]i and Vm measurements in particular have been published for skeletal muscle arterial endothelium15,16 as well as lymphatic vessel endothelium17. In addition to primary studies utilizing the endothelial tube approach, a comprehensive review of its advantages and disadvantages8 can be consulted to determine if this experimental tool is appropriate for a specific study. In brief, an advantage is that the key physiological components of endothelial cell function are retained (e.g., Ca2+ influx and intracellular release, hyperpolarization of Vm up to the Nernst potential for K+ via SKCa/IKCa activation, and endothelial intercellular coupling via gap junctions) without confounding factors such as perivascular nerve input, smooth muscle voltage-gated channel function and contractility, circulation of blood, and hormonal influences8. In contrast, commonly used cell culture approaches introduce significant alterations in morphology18 and ion channel expression19 in a manner that can greatly obfuscate comparisons to physiological observations determined ex vivo or in vivo. Limitations include a lack of integration with other essential components for regulating blood flow, such as smooth muscle and restricted flexibility in an experimental schedule, as this model is optimally tested within 4 h of intact vascular segment isolation from the animal.
Building from a previous video protocol authored by Socha and Segal12 and recent experimental developments in the interim6,15,16, we hereby demonstrate the isolation of fresh endothelium from posterior cerebral arteries and simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively. Additionally, this experiment entails continuous superfusion of salt solutions and pharmacological agents during physiological conditions (pH 7.4, 37 °C). We chose the posterior cerebral artery, as it yields isolated endothelium with the structural integrity (cells coupled through gap junctions) and sufficient dimensions (width ≥50 µm, length ≥300 µm) amenable for intra- and intercellular signaling along and among endothelial cells. In addition, studies of the rodent posterior cerebral artery are substantially represented in the literature and encompass examination of fundamental endothelial signaling mechanisms, vascular development/aging, and pathology20,21,22. This experimental application is expected to yield a high-throughput analysis of cerebral endothelial function (and dysfunction) and will thereby allow for significant advancements in the understanding of blood flow regulation throughout aging and the development of neurodegenerative disease.
Before conducting the following experiments, ensure that all animal care use and protocols are approved by the Institutional Animal Care and Use Committee (IACUC) and performed in accord with the National Research Council's "Guide for the Care and Use of Laboratory Animals" (8th Edition, 2011) and the ARRIVE guidelines. The IACUC of Loma Linda University has approved all protocols used for this manuscript for male and female C57BL/6 mice (age range: 3 to 30 mo).
1. Equipment and Materials
NOTE: Details of materials required for the protocol can be found in the Table of Materials, Reagents, and manuals or websites associated with the respective vendors.
2. Preparation of Solutions and Drugs
3. Dissection and Isolation of Cerebral Artery
NOTE: All dissection procedures require specimen magnification (up to 50x) via stereomicroscopes and illumination provided by fiber optic light sources. To perform dissection procedures for isolation of the brain and arteries, use sharpened dissection instruments. Microdissection tools to isolate and clean arteries include sharpened fine-tipped forceps and Vannas style dissection scissors (3 to 9.5 mm blades).
4. Preparation of Endothelial Tube and Superfusion
NOTE: The endothelial tube is prepared as described previously12, with modifications for the cerebral artery6.
5. Dye Load, Wash-Out, and Temperature Settings
6. Simultaneous Measurement of [Ca2+]i and Vm
7. Visualization of Cell-to-Cell Coupling
The schematic demonstration of the protocol described above is shown in the attached figures. A brain isolated from a young adult male C57BL/6N mouse (5 months) is shown in Figure 1A. Posterior cerebral arteries are carefully isolated from the Circle of Willis, removed without connective tissue, and cut into segments (Figure 1B-D). From partially digested arterial segments, the intact endothelial tube is produced...
In light of recent developments6,15,16,17, we now demonstrate the method to isolate mouse cerebral arterial endothelium in preparation for simultaneous measurement of [Ca2+]i and Vm underlying EDH consistently for ~2 h at 37 °C. Although technically difficult, we can measure cell-to-cell coupling as well (see reference6, Figure 1). I...
The authors declare no conflicts of interest.
We thank Charles Hewitt for excellent technical assistance while establishing equipment and supplies needed for the current protocols. We thank Drs. Sean M. Wilson and Christopher G. Wilson, from the LLU Center for Perinatal Biology, for providing us with an additional inverted microscope and electrometer, respectively. This research has been supported by National Institutes of Health grant R00-AG047198 (EJB) and Loma Linda University School of Medicine new faculty start-up funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Name | Company | Catalog Number | Comments |
Glucose | Sigma-Aldrich (St. Louis, MO, USA) | G7021 | |
NaCl | Sigma | S7653 | |
MgCl2 | Sigma | M2670 | |
CaCl2 | Sigma | 223506 | |
HEPES | Sigma | H4034 | |
KCl | Sigma | P9541 | |
NaOH | Sigma | S8045 | |
ATP | Sigma | A2383 | |
HCl | ThermoFisher Scientific (Pittsburgh, PA, USA) | A466250 | |
Collagenase (Type H Blend) | Sigma | C8051 | |
Dithioerythritol | Sigma | D8255 | |
Papain | Sigma | P4762 | |
Elastase | Sigma | E7885 | |
BSA | Sigma | A7906 | |
Propidium iodide | Sigma | P4170 | |
DMSO | Sigma | D8418 | |
Fura-2 AM dye | Invitrogen, Carlsbad, CA, USA | F14185 | |
Recirculating chiller (Isotemp 500LCU) | ThermoFisher Scientific | 13874647 | |
Plexiglas superfusion chamber | Warner Instruments, Camden, CT, USA | RC-27 | |
Glass coverslip bottom (2.4 × 5.0 cm) | ThermoFisher Scientific | 12-548-5M | |
Anodized aluminum platform (diameter: 7.8 cm) | Warner Instruments | PM6 or PH6 | |
Compact aluminum stage | Siskiyou, Grants Pass, OR, USA | 8090P | |
Micromanipulator | Siskiyou | MX10 | |
Stereomicroscopes | Zeiss, NY, USA | Stemi 2000 & 2000-C | |
Fiber optic light sources | Schott, Mainz, Germany & KL200, Zeiss | Fostec 8375 | |
Nikon inverted microscope | Nikon Instruments Inc, Melville, NY, USA | Ts2 | |
Phase contrast objectives | Nikon Instruments Inc | (Ph1 DL; 10X & 20X) | |
Fluorescent objectives | Nikon Instruments Inc | 20X (S-Fluor), and 40X (Plan Fluor) | |
Nikon inverted microscope | Nikon Instruments Inc | Eclipse TS100 | |
Microsyringe pump controller (Micro4 ) | World Precision Instruments (WPI), Sarasota, FL, USA | SYS-MICRO4 | |
Vibration isolation table | Technical Manufacturing, Peabody, MA, USA | Micro-g | |
Amplifiers | Molecular Devices, Sunnyvale, CA, USA | Axoclamp 2B & Axoclamp 900A | |
Headstages | Molecular Devices | HS-2A & HS-9A | |
Function generator | EZ Digital, Seoul, South Korea | FG-8002 | |
Data Acquision System | Molecular Devices, Sunnyvale, CA, USA | Digidata 1550A | |
Audible Baseline Monitors | Ampol US LLC, Sarasota, FL, USA | BM-A-TM | |
Digital Storage Oscilloscope | Tektronix, Beaverton, Oregon, USA | TDS 2024B | |
Fluorescence System Interface, ARC Lamp + Power Supply, Hyperswitch, PMT | Molecular Devices, Sunnyvale, CA, USA | IonOptix Systems | |
Temperature Controller | Warner Instruments | TC-344B or C | |
Inline Heater | Warner Instruments | SH- 27B | |
Valve Controller | Warner Instruments | VC-6 | |
Inline Flow Control Valve | Warner Instruments | FR-50 | |
Electronic Puller | Sutter Instruments, Novato, CA, USA | P-97 or P-1000 | |
Microforge | Narishige, East Meadow, NY, USA | MF-900 | |
Borosilicate Glass Tubes (Trituration) | World Precision Instruments (WPI), Sarasota, FL, USA | 1B100-4 | |
Borosilicate Glass Tubes (Pinning) | Warner Instruments | G150T-6 | |
Borosilicate Glass Tubes (Sharp Electrodes) | Warner Instruments | GC100F-10 | |
Syringe Filter (0.22 µm) | ThermoFisher Scientific | 722-2520 | |
Glass Petri Dish + Charcoal Sylgard | Living Systems Instrumentation, St. Albans City, VT, USA | DD-90-S-BLK | |
Vannas Style Scissors (3 mm & 9.5 mm) | World Precision Instruments | 555640S, 14364 | |
Scissors 3 & 7 mm blades | Fine Science Tools (or FST), Foster City, CA, USA | Moria MC52 & 15000-00 | |
Sharpened fine-tipped forceps | FST | Dumont #5 & Dumont #55 |
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